Clonal polymerase chain reaction–single-strand conformational polymorphism analysis: An effective approach for identifying cloned sequences

• Published in: Analytical biochemistry Vol. 378; no. 1; pp. 111 - 112
• Main Authors: Zhou, H., Hickford, J.G.H.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.07.2008
• Subjects: Alleles; Animals; Base Sequence; Cloning, Molecular - methods; Genome - genetics; Molecular Sequence Data; Polymerase Chain Reaction - methods; Polymorphism, Single-Stranded Conformational - genetics; Sequence Alignment; Sheep
• ISSN: 0003-2697; 1096-0309; 1096-0309
• DOI: 10.1016/j.ab.2008.04.005

The amplification of target sequences from genomic DNA can result in more than one amplicon sequence being produced even when highly specific primers are used. Here we present a clonal polymerase chain reaction–single-strand conformational polymorphism (PCR–SSCP) approach for screening cloned amplicons and identifying particular clones prior to sequence determination. Comparison of the PCR–SSCP patterns of the cloned amplicons with the PCR–SSCP patterns observed for the DNA templates from which the clones were derived allows PCR artifacts, different alleles, and even different loci to be differentiated prior to sequencing. Using this approach, the number of clones required for reliable sequence determination is minimized, and complex “mixed” amplicons can be resolved easily, cost-effectively, and reliably.